基于ITS与factor1-alpha序列桉树焦枯病菌的双重PCR快速检测

Duplex PCR technique based on the sequences of ITS and factor 1-alpha gene for the rapid detection of Calonectria morganii

桉树焦枯病是威胁桉树生长的首要病害,建立准确、有效的桉树焦枯病的PCR快速检测技术是桉树焦枯病前期诊断的必要手段。试验以桉树焦枯病原菌(Calonectria morganii)DNA为模板,分别以ITS和factor 1-alpha序列为靶区域,针对Calonectria属和C.morganii设计了CYS1/CYS2和EF-S-1/EF-A-1两对特异性引物,建立了基于属和种的双重PCR快速检测技术。利用引物CYS1/CYS2可以从全部Calonectria属的供试菌株中扩增出一条351bp大小的条带,单独用特异性引物EF-S-1/EF-A-1进行PCR扩增,仅病原菌扩增出197bp的条带,同时使用2对引物时,病原菌可扩增出两条明亮条带。当体系退火温度为53℃时,DNA灵敏度检测限度达到450fg/μL。野外田间时效检测结果显示,该体系能准确检测出不同发病程度桉树组织上的病原菌,完全符合田间检测的要求。这是关于桉树焦枯病快速检测的首次报道。

Eucalyptus dieback is the primary threat to the growth of eucalyptus.It is necessary to establish an ac-curate and effective PCR rapid detection technique for the early diagnosis of this disease.The duplex PCR tech-nique,for rapid detection of genera and species of eucalyptus dieback disease pathogen was established with the total DNA as templates,and two pairs of specific primers （CYS1/CYS2 and EF-S-1/EF-A-1 ）for Calonectria de Not and C .morganii based on their ITS sequence and factor 1-alpha gene were designed,respectively.The results showed that all tested strains belonging to Calonectria genera were amplified a product of 351 bp using primers CYS1/CYS2,while only C .morganii strains were amplified a product of 197 bp using primers EF-S-1/EF-A-1,and the duplex PCR with these two pairs of primers could amplify the two products.Furthermore,at the annealing temperature of 53 ℃,the minimum amount of detected DNA was 450 fg/μL.The infected eucalyptus tissue with different levels could be accurately detected by the duplex PCR technique,indicating that the duplex PCR technique fits entirely in the field test.This was the first report about the rapid detection technique on eucalyptus dieback caused by C .morganii .